The Effects Of PCV2 On NF-κB Signal In Piglet's Lymphocytes In Vitro

Porcine circovirus type 2 (PCV2), the primary pathogen of postweaning multisystemic wasting syndrome (PMWS), mainly infects the monocytes and macrophages, inducing loss of lymphocytes and the change of cytokines and receptors in piglets. NF-κB signal is one of important pathways to adjust inflammation reaction. The objective of the present study was to study and research the activation of NF-κB signal, the activated mechanism and the relationship between NF-κB signal and cytokines after PCV2 infection in vitro.Test one:Five conventional post-weaning piglets which were free of PCV2 and PRRSV antibody were chosen as the experiment animals. The cells isolated from spleens with pinhead of injectors were distributed into two groups (control group and PCV2 group). Lymphocytes were harvested after cells treated with or without PCV2 for 0,6,12,24h, respectively. The nuclear translocation of NF-κB was observed by confocal laser scanning microscope, and the protein levels of p65 in nucleus, phosphorylation-IκBαand phosphorylation-P38 in cytoplasm were detected by Western-Blot, and NF-κB DNA binding activity were detected by electrophoretic mobility shift assay (EMSA). The result of confocal laser scanning microscope showed that there was no nuclear translocation of NF-κB/P65 after lymphocytes incubated with 0h,6h and 12h except 24h in control group, however, nuclear translocation of NF-κB/P65 was observed after incubated with 6 hours in PCV2 group, and the amount of the nuclear translocation increased with the increased incubation time. The results of western blot and EMSA showed that NF-κB/P65,p-IκBα,p-P38 and the binding of NF-κB and DNA were no change after lymphocytes incubated with 0h,6h and 12h except 24h in control group, while the levels of NF-κB/P65 and p-IκBαand NF-κB DNA binding activity were detected obviously and increased with the incubation time in PCV2 group. After incubation with 24 h in PCV2 group, the NF-κB/P65 and p-IκBαand NF-κB DNA binding activity were significantly increased (P<0.05) compared with that in control group. The tendency of NF-κB/P65 was quite consistent with the tendency of p-IκBα, and the correlation of them was significantly positive correlation,whose coefficient correlation was 0.909. There were no obvious difference (P>0.05) in the protein levels of p-P38 in PCV2 group. The correlation analysis between NF-κB and cytokines expression which aquired in our previous results was made. The results manifested that NF-κB was significantly positive correlation with TNF-α,IL-6RmRNA and IL-10mRNA, whose coefficient correlation was got at 0.757(P=0.002),0.783(P=0.000),0.764(P=0.000) respectively, but no obvious correlation with IL-6,IL-6mRNA,IL-10 and IL-lOmRNA. The consequence maybe resulted from not in one trail, but on the other point it indicated that PCV2 could modulate the expression and transcription of cytokines by NF-κB signal.In summary, all above results demonstrated PCV2 can activated the NF-κB signal including translocation to the nuclear and binding with DNA, then inducing the transcription and expression of inflammatory cytokines, but seemly there is no obvious relevant to P38 signal pathway.Test two:Five conventional post-weaning piglets which were free of PCV2 and PRRSV antibody were chosen as the experiment animals. The cells isolated from spleens with pinhead of injectors were distributed into four groups which treated with PCV2 for 0,6,12,4h, respectively. All lymphocytes of four groups were harvested after 24hours. The protein levels of NF-κB/P65, p-IκBαand p-P38 were detected by Western-Blot and NF-κB DNA binding activity was detected by EMSA. The results showed that the protein levels of NF-κB/P65 of lymphocytes incubated with 24 h was extremely significantly increased (P<0.01) compared with Oh and 6h, and which also significantly increased compared with 12h. The result of NF-κB DNA binding activity was same to NF-κB/P65 protein. The protein levels of p-IκBαwas highest in 24HPI, which was extremely notably (P<0.01)compared with Oh and 6h. The two among the other groups has notably difference (P<0.05)except 6h and 12h. The protein level of p-P38 has a little decrease at 6h and rise at 12h and 24h, that the p-P38 at 24h was significantly increased compared with 6h. All the results demonstrated PCV2 can activated the NF-κB signal which could modulate the related pro-inflammation cytokines.